In vitro comparative evaluations of the postantifungal effect: synergistic interaction between flucytosine and fluconazole against Candida albicans

In vitro comparative evaluations were performed to study the efficacy of combinations of flucytosine and fluconazole in producing a postantifungal effect (PAFE) on Candida albicans. Initial studies were done to determine MIC, FIC (fractional inhibitory concentration) and optimal PAFE parameters. A turbidometric method was used to measure yeast cell growth following exposure to different concentrations of the two drugs for periods of 0.5, 1 or 2 h at temperatures of 30 degrees C and 37 degrees C. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. Ten strains of C. albicans were then assayed (30 degrees C; 2 h exposure time) and a synergistic PAFE was evidenced with the two drugs at concentrations well below their individual MICs. PAFEs ranging from 3.8 to 10.5 h, which persisted for 1.2-2.5 h longer than those achieved with either agent separately, were evidenced when flucytosine and fluconazole were combined (flucytosine: fluconazole ratios of 1:16-1:32) at concentrations ranging from 0.024 to 0.098 micrograms ml-1 and 0.78 to 1.56 micrograms ml-1 respectively. The concentrations of each agent required to produce an optimal PAFE varied according to the C. albicans strain being assayed.     

Characterization of Candida albicans Strains Found in Patients of Three Intensive Care Units

Summary:  In three intensive care units (surgery, neurosurgery, neurology) a total of 225 patients were examined in weekly intervals over a period of three months for colonization with C. albicans on the oral mucosa and the perianal region. 155 C. albicans isolates were identified by means of their properties of lipase and proteinase production and resistance to 5-fluorocytosine (5-FC). 42% of all patients harboured C. albicans (oral mucosa 38 %, perianal region 9 %). 96.1 % of the strains showed production of lipase, 67.1 % production of proteinase and 29.7 % resistance to 5-FC. Mentionable in comparison with other studies is the high percentage of 5-FC resistant strains of C. albicans , as well as the increasing frequency of asymptomatic infection with 5-FC resistant C. albicans strains which increases with the time of hospitalization.
Zusammenfassung:  Über einen Zeitraum von 3 Monaten wurden in wöchentlichen Abständen Mundschleimhaut und Peria-nalregion von insgesamt 225 Patienten dreier Intensivstationen (Chirurgie, Neu-rochirurgie, Neurologie) auf C. albicans -Besiedelung untersucht. Dabei wurden 155 C. albicans -Isolate identifiziert und mittels der Stammeigenschaften Lipase-bildung, Proteinasebildung und 5-Fluoro-cytosin-(5-FC)-Resistenz differenziert. 42 % aller Intensivpatienten erwiesen sich als mit C. albicans besiedelt (Mundschleimhaut 38 %, Perianalregion 9 %). 91.1 % der Stämme zeigten Lipasebildung, 67.1 % Proteinasebildung und 29.7 % Resi-stenz gegenüber 5-FC. Auffallend war der im Vergleich zu anderen Kollektiven hohe Prozentsatz an 5-FC-resistenten C. albicans -Stämmen und eine mit zunehmender Liegedauer auf Intensivstation ansteigen-de Besiedelungsfrequenz mt 5-FC-resistenten C. albicans -Stämmen.     

Fungistatic Activity of Miconazole against Candida albicans in Relation to pH and Concentration of Nonprotonated Drug

At less than 10(-5) M, miconazole (MCZ) exerts a fungistatic effect on Candida albicans, presumably by interfering with ergosterol biosynthesis. The imidazole moiety of MCZ is subject to protonation (pKa approximately 6.5). Based on pKa and greater water solubility of protonated (MCZH+) versus nonprotonated (MCZo) drug, fungistatic action ought to be markedly affected by environmental pH, but apparently it is not. In this report growth phase, pH, and concentrations of MCZ and MCZo have been studied in relation to fungistasis. Yeasts were grown in a synthetic liquid medium and MCZ effects were monitored by viability determinations. Results showed that fungistatic activity is little affected by growth phase and is largely independent of drug concentration and pH. Antagonism of fungistasis by low pH was demonstrated only at less than 10(-7) M MCZ. Data supported earlier proposals that MCZo is required for biological activity and suggested that target sites are saturated at very low levels of drug.     

Intracellular killing of Candida albicans by human neutrophils is potentiated by exposure to combinations of amphotericin B and 5-fluorocytosine

Combination treatment with amphotericin B and 5-fluorocytosine is synergistic and has become clinically useful in the treatment of various forms of systemic candidosis. The synergy between these two compounds may be explained in part by their combined effect on the interaction between fungal cells and host phagocytes. Pretreatment of Candida albicans for 2 h with either amphotericin B or 5-fluorocytosine or the two agents in combination did not inhibit or enhance phagocytosis by glass-adherent human neutrophils (P greater than 0.05). Intracellular killing of pretreated yeast cells was not influenced by antifungals alone (P greater than 0.05), but pretreatment of C. albicans with 5.0 mg l-1 amphotericin B + 10 l-1 5-fluorocytosine or 5.0 mg l-1 amphotericin B + 50 mg l-1 5-fluorocytosine significantly enhanced the ability of neutrophils to kill the number of viable yeast cells intracellularly (P less than 0.001).     

The Effect of Antimycotics on Secretory Acid Proteinase of Candida albicans

Summary

The effect of antimycotics on secretory aspartate (acid) proteinase, a virulence enzyme of Candida albicans, was investigated.

The conditions of the study were such as to induce proteinase production in the stationary phase of growth (25-40 hours), when no antifungal tested, except the polyene derivative methyl partricin, significantly reduced the viability of the culture.

Among azole derivatives, fenticonazole (FZ) but not miconazole, fluconazole or ketoconazole, exerted strong inhibition on proteinase, in typical dose-diphasic pattern, (0.01 μg/ml; 1-10 μg/ml). 5-fluorocytosine (5-FC) was also inhibitory at a dose interval 1-10 μg/ml. In all cases, the inhibition concerned the synthesis of the enzyme rather that its activity as suggested by the results of comparative ELISA, SDS-PAGE and spectrophotometric methods of proteinase detection.

Finally, the inhibition of proteinase production by FZ and 5-FC mainly reflected the effect of these antimycotics on general protein synthesis.     

Comparison of 12 liquid media for germ tube production of Candida albicans and C. tropicalis

Infections caused by yeast of the genus Candida are the most common fungal infections, being Candida albicans the most common isolated species among them. The rapid identification of this yeast is mostly based on the production of germ tube in human or animal serum. This study describes the use of 12 different liquid media for germ tube production at 2, 2.5, 3 and 4 h. We examined 193 yeasts, including 157 (81.3%) C. albicans and 36 (18.7%) Candida tropicalis for the production of germ tube. The germ tube production of C. albicans was mostly observed in human serum (98%) followed by rabbit serum (89.8%), brain heart infusion broth (84%) and sheep serum (74.5%) at 2 h. An incubation time exceeding 2 h i.e. 2.5 h or later, C. tropicalis strains were observed to produce germ tubes. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for germ tube production of human serum at 2 h were 98%, 100%, 100% and 92.3% respectively. In all tested sera, an incubation period of more than 2 h improves the sensitivity, but decreases the specificity as well as PPV and NPV of germ tube test (GTT). In conclusion, human serum was observed to be the most appropriate medium to be preferred for GTT, with an incubation period of 2 h.     

In vitro and in vivo activity of antifungal agents in combination with fleroxacin, a new quinolone

The in vitro and in vivo interaction of fleroxacin with amphotericin B (Amph B), flucytosine (5-FC) and azoles against Candida albicans strains was tested. In vitro the interaction between fleroxacin and various antifungals was not dependent on the incubation time. Fleroxacin neither enhances nor antagonizes the in vitro activity of Amph B at high concentration (50-100 micrograms/ml). Fleroxacin has a synergistic effect with ketoconazole (KETO), but this is not observed with itraconazole (ITRA) or fluconazole (FLU). In no instance antagonism was observed. The activity of 5-FC was antagonized by fleroxacin being generally reduced by 2-4 dilution steps. In murine candidosis the efficacies of all antifungal drugs were not influenced by addition of 100 mg/kg fleroxacin. Therefore, the effects seen in in vitro tests are most probably not relevant for the clinical use of a combination of fleroxacin with antifungal drugs.     

Modification of responses of Candida albicans to cisplatin by membrane damaging antimycotic agents

The genotoxic, antineoplastic platinum coordination complex, cisplatin (cis-diamminedichloroplatinum (II], exists as a positively charged aquated complex in water solution and as a neutral, nonaquated complex in saline solution. Candida albicans exhibited greater susceptibilities to cellular inactivation and induction of mitotic recombination when treated with the aquated rather than the nonaquated drug. The differential in responses was expressed by cells grown after treatment at 37 degrees C or at 25 degrees C, a temperature which promotes recovery from DNA damages by the yeast generally. Studies with protoplasts established that cell wall components do not influence cellular reactions to either form of the drug. However, membrane damaging antimycotic agents markedly affected responses. Pretreatments with fungistatic ketoconazole or with miconazole, under fungistatic or fungicidal conditions, enhanced cellular resistance to inactivation by aquated cisplatin: the effect was more pronounced with post-cisplatin growth at 25 degrees C than 37 degrees C. Fungicidal pretreatments with amphotericin B or miconazole greatly increased susceptibilities of surviving cells to the lethal and recombinagenic effects of nonaquated cisplatin with post-cisplatin recovery at 25 degrees C or 37 degrees C. Possible mechanisms underlying these responses and their implications for stability of C. albicans populations in cancer patients undergoing therapy with cisplatin are discussed.     

Reassessment of the in vitro synergistic effect of fluconazole with the non-steroidal anti-inflammatory agent ibuprofen against Candida albicans

We reassessed the in vitro synergistic effect of fluconazole with the non-steroidal anti-inflammatory agent ibuprofen against the pathogenic yeast Candida albicans. No synergistic effect of fluconazole combined with ibuprofen was seen against fluconazole-susceptible strains, but a remarkable effect was seen against fluconazole-resistant strains (FIX index: 0.02-0.03). Furthermore, vigorous growth of the microorganism, the so-called 'Eagle effect', was observed at concentrations higher than the minimal inhibitory concentrations of ibuprofen and fluconazole. Our results suggest that the combination of ibuprofen and fluconazole should prove useful for treating infection caused by fluconazole-resistant C. albicans.     

In vitro postantifungal effect,adhesion traits and haemolysin production of Candida dubliniensis isolates following exposure to 5‐fluorocytosine

The phenomenon of postantifungal effect (PAFE), which is the suppression of candidal growth following brief exposure to antifungal agents, is linked with candidal pathogenicity. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation and relative cell surface hydrophobicity (CSH) are all adhesion traits of candidal pathogenicity. Ability to produce haemolysin by Candida species is also a determinant of its pathogenicity. There is no information on either the PAFE or its impact on adhesion traits and haemolysin production of oral Candida dubliniensis isolates following exposure to 5‐fluorocytosine (5‐FC). Hence, the focus of this investigation was to research the in vitro PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production on 20 C. dubliniensis isolates following exposure to 5‐FC. Following obtaining the minimum inhibitory concentration (MIC) of 5‐FC, isolates of C. dubliniensis were exposed to sub‐lethal concentrations (×3 MIC) of 5‐FC for 1 h. After this brief exposure, the antimycotic was removed and PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production was determined by formerly described in vitro methods. MIC (μg/ml) of C. dubliniensis isolates to 5‐FC ranged from 0.002 to 0.125. The mean PAFE (hours) elicited by 5‐FC on C. dubliniensis isolates was approximately 1 h. Exposure to 5‐FC suppressed the ability of C. dubliniensis isolates to adhere BEC, GT formation, relative CSH and haemolysin activity by a mean percentage reduction in 50.98%, 29.51%, 36.79% and 12.75% (P < 0.001 for all) respectively. Therefore, brief exposure of C. dubliniensis isolates to 5‐FC appears to exert an antifungal effect by subduing its growth, adhesion traits as well as haemolysin production.     

Evaluation of Mueller-Hinton-agar as a simple medium for the germ tube production of Candida albicans and Candida dubliniensis

Candida albicans is the most frequently isolated yeast species from clinical specimens. A classical rapid presumptive differentiation from non- albicans species is based on its ability to produce germ tubes after incubation in human serum. The only non- albicans Candida species producing germ tubes is Candida dubliniensis. In this study, we evaluated Mueller-Hinton-agar (MH-agar) as a medium for germ tube formation of C. albicans and C. dubliniensis . A total of 859 yeast isolates from stool samples, including 632 strains of C. albicans , 10 C. dubliniensis and 217 other yeast strains from 20 different species, were grown on Sabouraud glucose (2%) agar at 37 °C for 24–72 h. Species were identified by standard methods. For the germ tube test (GTT), an inoculum from a single colony was streaked onto a MH-agar plate and covered by a sterile coverslip. After incubation at 37 °C for 2 h, the MH plates were examined using a light microscope at ×200. The GTT was positive in 578 of 632 C. albicans strains (sensitivity 91.5%), in six of 10 C. dubliniensis strains (sensitivity 60.0%), and in none of the other yeast strains. MH-agar is a suitable medium for the GTT and the presumptive identification of C. albicans . It is safer to use than human serum and is widely available in microbiology laboratories.     

Changes in Candida albicans colonization and morphology under influence of voriconazole

The aim was the investigation of fungal colonization and morphological alterations under the influence of voriconazole in an in vitro system. Voriconazole stopped growth and colonization of Candida albicans (wild type SC5314) on cover slips in microtiter plates dependent on drug concentration, the time of Candida growth before the input of voriconazole and oxygen concentration. The direct microscopy by fluorescence staining with the optical brightener Blancophor showed short bizarrely deformed mycelia looking swollen. The colonization on cover glass was diminished. Microcolonies or starting of biofilm formation as in the control was not observed. The metabolic activity was demonstrated by vital staining with FUN 1 resulting in red fluorescent structures in the yeast forms and mycelia in the controls. Under voriconazole influence the remaining cells only showed a green or pale yellow fluorescence. Most of the cells lost their metabolic activity.     

A rapid Candida albicans hyphal-form growth inhibition assay: determination of myelomonocytic-mediated antifungal activity

An in vitro microassay for the measurement of Candida albicans hyphal-form growth inhibition by myelomonocytic cells is described. The assay is rapid, easy-to-perform and objective. A Candida strain capable of in vitro dimorphic transition from yeast to hyphal form has been employed. The assay is based on the incorporation of 3H-glucose by the fungus, the effect being dependent upon the time of pulse, size of the inoculum and concentration of radiolabelled metabolite. In particular, C. albicans hyphal form, obtained by a 3 h incubation in vitro in the presence of 10% fetal calf serum, is co-incubated with the effector cells. A pulse with 3H-glucose in water is then performed and the radioactivity incorporated by the residual Candida is taken as an indication of hyphal growth. We found that polymorphonuclear cells, peritoneal macrophages and the cloned GG2EE macrophage cell line significantly inhibited hyphal growth, the effects being time and effector-to-target cell ratio dependent.     

Effect of Amorolfine (Ro 14–4767/002) on in vitro Phagocytosis and Intracellular Killing of Cartdida albicans by Human Neutrophils: Die Wirkung von Amorolfin Po 14–4767/002) in vitro auf Phagozytose und intrazellulare Abtotung von Candida albicans durch menschlische Neutrophile

The effect of amorolfine (Ro 14-4767/002) on phagocytosis and intracellular killing of Candida albicans blastospores was determined in human neutrophil monolayer assays. At 0.2, 2 and 5 micrograms/ml amorolfine did not have any significant inhibitory or enhancing effect on phagocytosis whether following simultaneous addition of blastospores and drug to the neutrophils, prior treatment of neutrophils for 2 h before addition of blastospores or prior treatment of blastospores for 2 h. Simultaneous addition of amorolfine resulted in a significant increase in killing at all concentrations. This increase was not significantly enhanced by either preincubation of neutrophils or blastospores for 2 h with the drug.     

An ex-vivo oral mucosa infection model for the evaluation of the topical activity of antifungal agents

Although Nystatin has been used since 1950s as a non-absorbable antifungal agent, there is still no reliable in-vivo data available stating a dose-effect relationship of Nystatin-suspension in the treatment of oropharyngeal infection with Candida albicans. Here, we studied the efficacy of a commercially available topical Nystatin suspension in a new ex-vivo model of candidiasis using porcine oral mucosa. After 48 and 96 h of C. albicans infection, 230 IU Nystatin (standard dosage), 100 IU and 20 IU proved to be equally efficacious. Multiple applications of Nystatin were not superior compared with single application. In dosages of 10 and 0.1 IU the activity of Nystatin suspension against C. albicans was no longer confirmed. In an agar diffusion model, the minimal biocidal concentration of Nystatin proved to be 0.25 IU. Our results suggest that the proposed porcine ex-vivo model is much closer to the in-vivo situation compared with other established in-vitro models of the treatment of muco-cutaneous candidiasis and may provide a substitute for animal models in the investigation of antifungal agents. Additionally, it seems to be a valuable tool for further investigations of the pathogenesis of C. albicans infections.     

Alterations Produced by Sertaconazole on the Morphology and Ultrastructure of Candida albicans: Sertaconazol-bedingte Veranderungen der Morphologie und Ultrastruktur von Candida albicans

We studied the changes produced in Candida albicans yeast cells after treatment in vitro with sertaconazole, with light microscopy, transmission and scanning electron microscopy. Four different concentrations were evaluated (10(-6) M, 10(-5) M, 10(-4) M, 10(-3) M) and two times of action (12 and 17 h) for each concentration. This new antimycotic agent shows a considerable destructive effect in C. albicans cells. The alterations produced by sertaconazole were already manifested after 12 h at the concentration of 10(-6) M and a strong fungicidal activity recorded at 10(-4) M.     

Four cases of Candida albicans infections with isolates developing pink colonies on CHROMagar Candida plates

Candida albicans, the most commonly isolated yeast species, is typically identified by its green colony-colour on CHROMagar Candida plates. We here report four cases of Candida albicans infections, in which the initial identification was non-albicans isolates due to a clear pink colour of the colonies on CHROMagar Candida plates. However, classical phenotypic criteria, biochemical assimilation pattern and molecular characterisation identified all four isolates as C. albicans isolates.     

The Influence of Some Antifungal Drugs on In Vitro Adherence of Candida albicans to Human Buccal Epithelial Cells

Summary:  An in vitro adherence test with 10 Candida albicans strains to buccal epithelial cells (BEC) was performed in a medium with the following antifungal drugs: nystatin, amphotericin B, 5-fluorocytosine, clotrimazole and ketoconazole. Simultaneously, an in vitro adherence test was made without drugs added, but the fungal cells had been previously treated with the same drugs. All antifungal drugs applied significantly inhibited the adherence of C. albicans to BEC (p < 0.01). Pretreatment of the fungi with drugs inhibited their adherence to BEC stronger than the addition of the drugs to the test medium. Subinhibitory doses of the drugs were less effective than therapeutic ones. The most effective inhibition of the adherence was obtained with 5-fluorocytosine and ketoconazole, while nystatin turned out to be the least effective.
Zusammenfassung:  Mit 10 Candida albicans- Stämmen wurden Adhärenzteste in vitro mit Epithelzellen der Mundschleimhaut (BEC) unter Zusatz folgender Antimyzetika durchgeführt: Nystatin, Amphotericin B, 5-Fluorcytosin, Clotrimazol und Ketoconazol. Parallel dazu wurden Adhärenzteste ohne Antimyzetika-Zusatz, jedoch nach antimyzetischer Vorbehandlung der Pilzzellen durchgeführt. Alle verwendeten Antimyzetika hemmten signifikant die Adhärenz von C. albicans an BEC (p < 0.01). Die antimyzetische Vorbehandlung hemmte die Adhärenz stärker als der Antimyzetika-Zusatz während des Adhärenzversuches. Subinhibitorische Dosen waren weniger wirksam als therapeutische Dosen. Die stärkste Adherenzhemmung wurde bei 5-FC und Ketoconazol, die schwächste bei Nystatin beobachtet.     

Effect of licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth and virulence properties of Candida albicans

Candida albicans is the predominant causal agent of candidiasis. Its ability to form hyphae and biofilm has been suggested to be key virulence factors. In this study, we investigated the effect of major licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth, biofilm formation and yeast-hyphal transition of C. albicans. The synergistic effect of licorice compounds with the antifungal drug nystatin was also evaluated. Minimal inhibitory concentrations (MICs) for C. albicans were determined using a microplate dilution assay. The synergistic effect with nystatin was determined similarly. The effect of licorice compounds on biofilm formation was evaluated using a microplate assay and crystal violet staining. The effect of licorice compounds on yeast-hyphal transition was determined by microscopic observation. The toxicity of licorice compounds towards oral epithelial cells was evaluated with an MTT assay. Glabridin and licochalcone A showed antifungal activity on C. albicans while glycyrrhizic acid had no effect. Complete growth inhibition occurred with sub-inhibitory concentrations of nystatin with either glabridin or licochalcone A. Biofilm formation was inhibited by 35-60% in the presence of licochalcone A (0.2 μg ml(-1)). A strong inhibitory effect (>80%) on hyphal formation was observed with licochalcone A or glabridin (100 μg ml(-1)). Glabridin and licochalcone A at high concentrations showed toxicity towards oral epithelial cells. In summary, glabridin and licochalcone A are potent antifungal agents and may act in synergy with nystatin to inhibit growth of C. albicans. Licochalcone A has a significant effect on biofilm formation, while both licochalcone A and glabridin prevented yeast-hyphal transition in C. albicans. These results suggest a therapeutic potential of licochalcone A and glabridin for C. albicans oral infections.     

Comparison of polar lipids from yeast and mycelial forms of Candida albicans and Candida dubliniensis

The aim of the present study was to compare polar lipids of yeast and mycelial forms of both Candida albicans and Candida dubliniensis. Cultures were harvested from Lee's medium after incubation at 37 degrees C for 48 h. Yeast and mycelial forms were washed, separated from one another, dried and lipids extracted and prepared for fast atom bombardment mass spectrometry analysis in the negative-ion mode. For fatty acids, differences between the yeast and mycelial forms were greater for C. dubliniensis than for C. albicans. For the phospholipid families, phosphatidic acid and phosphatidylethanolamine, differences between yeast and mycelial forms were greater for C. dubliniensis than for C. albicans. For both species, it is concluded that both fatty acid and phospholipid molecular species compositions differ according to whether the cells are in the yeast or mycelial form.